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Metagenomic Analysis of Soil
Introduction Soil harbors the largest microbial diversity per unit mass or volume (1). One gram of cultivated soil or grassland contains an estimated 2x10^9 prokaryotic cells and one gram of forest soil contains and estimated 4x10^7 prokaryotic cells. The heterogeneity of the soil itself is due to the complex chemical and biological properties of soil environments. (2) Soil is one of the most challenging of natural environments to study for microbiologists. This is because only a fraction of the soil microorganisms can be cultivated; therefore conventional studies of soil miss many organisms. Traditional microbiology approaches revealed that soils commonly harbor a broad array of antibiotic-related functions, some of which have been associated with the suppression of plant pathogens. So there is great interest in studying soil to explore genes that encode function of interest to biotechnology. Molecular methods can be used to isolate and analyze DNA extracted from soil samples without cultivation of microorganisms. The microbial DNA isolated from a soil sample represents the collective DNA of all the indegenous soil microorgansisms, and is named the soil metagenome. Using genomic DNA extracted from soil bypasses growing organisms and provides information on the collective soil meta-genome. Using this approach Dr VanElsas studied the metagenome of disease-suppresive soils.(3) Construction of soil DNA libraries Construction soil-based libraries involves the same methods as cloning of genomic DNA of individual microorganism. It includes extraction of DNA from the soil, fragmentation of the soil DNA by restriction-enzyme digestion, insertion of DNA fragments into an appropriate vector system, and transformation of the recombinant vectors into a host. Although the generation of soil libraries is conceptually simple, the size of the soil metagenome and the large number of clones that are required for full coverage make this a daunting task. The diagram shows the main steps in the construction of a metagenomic DNA library from a soil sample. Soil DNA is recovered through separation of cells from soil particles followed by cell lysis and DNA extraction, or by direct lysis of organisms contained in the soil and then DNA extraction. The DNA is then cut with restriction endonucleases, ligated into a cloning vector that can be a plasmid, BAC (bacterial artificial chromosome). Then the recombinant vector is introduced into a host and the library is grown. (4) Library analysis The metagenomic library can be analyzed by massive sequencing like next generation sequencing, NGS and comparing data to known databases or by function screens in which the library is screened from genes that have a particular function. In the example below the library is screened for clones with proteolytic activity. E.coli clones are detected on agar media containing skim milk by zones of clearance around the colonies. Results These approaches were used to clone genes from soil that code for lipases, proteases, oxidoreductase, B-glucosidases, amylases, antibiotics, antibiotic resistance enzymes and membrane proteins. The genes identified using function screens had little homology to known genes, which illustrates the enormous potential of soil-based metagenomic libraries.(4) Metabolomics has been used to identify microorganisms useful for bioremediation of enviromental contaminants such as bacteria with biphenyl dioxygenase genes that can grow in PCB-contaminated river sediment. Also bacteria containing enzymes that can degrade organic contaminants were identified. (4) Two interesting examples of new finding using metagenomic studies are the studies done by Mc Neal and collaborators and by Brady and collaborators. The first group prepared and then screened a BAC library from DNA extracted from uncultivated soil from New England; they found a clone that expressed a molecule related to indirubin an antileukemic drug (5). Dr. Brady's group discovered a violacein gene cluster in a cosmid library prepared with DNA from Ithaca, NY soil. Violacein is active against Gramp -positive bacteria and induces apoptosis in fibroblast cells(6). Conclusions The use of metagenomics in soil analysis allowed the characterization of microbial communities by random sequencing and by function screens. It is expected that more discoveries will be made in the future because of improvements in sequencing technologies and bioinformatic tools for analysis of the enormous amount of data produced. Also monitoring microbial activity through protein arrays and proteomics will allow more efficeint screening of metagenomic libraries. Soil microorganisms may be an almost unlimited resource of new genes encoding useful products, the use of metagenomic technology will be an important tool to discover them. References 1 Gans, J. et al. Computational improvements reveal great bacterial diversity and high metal toxicity in soil. 2005. Science 309, 1387–1390 2. http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi 3. Van Elsas JD et al. The metagenomics of disease suppressive soils – experiences from the METACONTROL project. 2008 Trends in Biotechnology Vol.26 No.11. 591-601. doi:10.1016/j.tibtech.2008.07.004 4. Daniel R. The metagenomics of soil. 2005. Nat. Rev. Microbiol. 3, 470-478. doi:10.1038/nrmicro1160 5. MacNeil, I.A. et al. (2001) Expression and isolation of antimicrobial small molecules from soil DNA libraries. J. Mol. Microbiol. Biotechnol. 3, 301–308 6. Brady, S.F. et al. (2001) Cloning and heterologous expression of a natural product biosynthetic gene cluster from eDNA. Org. Lett. 3, 1981–1984